Oral delivery of biologically active substances bound to vitamin B12

ABSTRACT

Complex of a drug, hormone, bio-active peptide, or immunogen with the carrier molecule vitamin B12 and a method for delivering the complex to the intestine of a host vertebrate in order to deliver the complex to the circulation of the host and thereby elicit a pharmacological response to the drug, hormone, bio-active molecule or to elicit a systemic immune response to the immunogen. The invention also provides a method for the production of the complex. Further, the invention provides medicaments containing the complex.

This application is a divisional, of application Ser. No. 08/061,343,filed May 17, 1993 now U.S. Pat. No. 5,428,023; which is a continuationof application Ser. No. 07/759,697 filed Sep. 9, 1991, now abandoned;which is a continuation of application Ser. No. 07/600,137 filed Oct.19, 1990 now abandoned; which is a continuation of application Ser. No.07/084,821 filed Jun. 9, 1987, now abandoned; which was filed under 35U.S.C. ♂371 based upon International application PCT/AU86/00299 filedOct. 10, 1986.

TECHNICAL FIELD

The present invention relates to oral delivery systems. Moreparticularly the invention relates to enhancing the absorption of activesubstances by administering these substances bound to vitamin B12 (VB12)or an analogue thereof.

BACKGROUND ART

The oral route of administration is perhaps the most preferable means ofdelivering an antigen or pharmaceutically active agent to man. Thisroute does however suffer from the major disadvantage that there isgenerally poor uptake of antigens or pharmaceutically active agents bythe gastrointestinal tract and some agents may be destroyed by prolongedexposure to proteolytic enzymes. In this regard, attemps to orallyimmunize man or animals in the past have met with limited success.Effective vaccination has generally only been achieved by theadministration of large quantities of antigen or by combining parenteralpriming with oral boosting. Recent work by us utilizing a number ofmolecules with the ability to bind to the intestinal mucosa hasdemonstrated effective oral immunization using low doses of thesebinding proteins or by coupling various antigens or haptens to thesecarriers. Uptake and delivery to the circulation of these molecules fromthe intestine seemed to be due to receptor mediated endocytosis.

It has been known for some time that a number of specific uptakemechanisms exist in the gut for uptake of dietary molecules. Thus thereare specific uptake mechanisms for monosaccharides, disaccharides, aminoacids and vitamins. Most of these uptake mechanisms depend upon thepresence of a specific protein or enzyme such as monosaccharidase ordisaccharidase situated in the mucosal lamina propria which binds to themolecule and transports it into the cells lining and lamina propria.

Two notable exceptions to these uptake mechanisms are found with irontransport and VB12 uptake. In both these cases a specific bindingprotein is released into the intestine, which binds to its ligand in thelumen of the gut.

Thus, during iron uptake in the intestine transferrin is released fromthe stomach, binds to iron and is in turn bound by a receptor on theduodenal mucosa. The receptor-transferrin-iron complex is then taken upby receptor mediated endocytosis.

Similarly, the absorption of physiological amounts of VB12 by the gutrequires that it be complexed with a naturally occurring transportprotein known as intrinsic factor (IF) (1-5). This protein is releasedinto the lumen of the stomach by parietal cells in the fundus. Oncebound to intrinsic factor, the VB12.IF complex interacts with a membranebound receptor for IF located on the terminal ileum of the smallintestine. The receptor-IF-VB12 complex is then internalized by aprocess of receptor mediated endocytosis (RME). Allen and Majerus (7)demonstrated that it is possible to chemically modify VB12, couple it toa resin and use the VB12-resin to affinity purify IF. This findingsuggested to us that it may be possible to couple large macromolecules(such as the resin used by Allen and Majerus) to VB12 and to stillpreserve it's ability to interact specifically with intrinsic factor. Bycoupling molecules to VB12 in such a way as to preserve the ability ofVB12 to interact with intrinsic factor it was hoped that we could usethe natural uptake mechanism for VB12, to deliver various proteins,drugs or other pharmaceutically active molecules to the circulation.

It is thus the object of this invention to utilize the VB12 uptakemechanism to transport active substances such a drugs, hormones,antigenic material and the like, covalently coupled to VB12 or ananalogue thereof, from the intestinal lumen into the circulation.

DISCLOSURE OF THE INVENTION

In a first embodiment the invention provides a complex which comprisesat least one active substance linked to at least one carrier moleculewhich is VB12 or an analogue thereof wherein the ability of the carrierto undergo the binding reactions necessary for uptake and transport ofVB12 in a vertebrate host and the activity of the active substance aresubstantially maintained.

In the context of the present invention, the term active substanceincludes all, part, an analogue, homologue, derivative or combinationthereof, of a hormone, bio-active peptide, therapeutic agent, antigen orhapten.

Preferred active substances for delivery according to the inventioninclude: hormones and bioactive peptides such as LHRH, insulin,testosterone, interferon, PMSG, HCG and inhibin; therapeutic agents suchas neomycin, salbutamol, pyrimethamine, penicillin G, methicillin,carbenicillin, pethidine, xylazine, ketamine hydrochloride, mephenesinand iron dextran; antigens or haptens including allergens, proteins,polysaccharides and secretory products such as grass pollens (forinstance barley and couch), weed pollens (e.g. clover, dock) treepollens (e.g. ash, cyprus), plant pollens (e.g. broom), epithelia (e.g.cat hair, dog hair, pig hair) and house dust mite, wheat chaff andkapok; a protein derived from or immunogens against influenza, measles,Rubella, smallpox, yellow fever, diphtheria, tetanus, cholera, plague,typhus, BCG, tuberculosis causing agents, Haemophilus influenzae,Neisseria catarrhalis, Klebsiella pneumoniae, pneumococci, streptococci;a secretory product derived from diphtheria, tetanus, cholera, plague,typhus, tuberculosis causing agents, Haemophilus influenzae, Neisseriacatarrhalis, Klebsiella pneumoniae, pneumococci, streptococci.,Streptococcus mutans, or is derived from a malarial parasite or thecausitive agent of coccidiosis in chickens.

Preferred analogues of VB12 include cyanocobalamin (CN--Cbl),aquocobalamin, adenosylcobalamin, methylcobalamin, hydroxycobalamin,cyanocobalamin carbanalide, and 5-o-methylbenzylcobalmin(5-OMeBza)CN--Cbl! as well as the desdimethyl, monoethylamide and themethylamide analogues of all of the above. Also included are the variousanalogues and homologues of cobamamide such as coenzyme B12 and5'-deoxyadencsylcobalamin. Other analogues include chlorocobalamin,sulfitocobalamin, nitrocobalamin, thiocyanatocobalamin, benzimidazolederivatives such as: 5,6-dichloro-benzimidazole, 5-hydroxybenzimidazole,trimethylbenzimidazole, as well as adenosylcyanocobalamin (Ade)CN--Cbl!,cobalamin lactone, cobalamin lactam and the anilide, ethylamide,monocarboxylic and dicarboxylic acid derivatives of VB12 or itsanalogues.

Preferred derivatives of VB12 include the mono-, di- and tricarboxylicacid derivatives or the proprionamide derivatives of VB12. Carriers mayalso include analogues of VB12 in which the cobalt is replaced by zincor nickel. The corrin ring of VB12 or its analogues may also besubstituted with any substituent which does not effect its binding toIF.

In a preferred embodiment of the invention there is provided a complexcomprising the lys-6 form of LHRH and VB12.

The complexes of this invention, of coupled active substances can beused to deliver these substances to any uni or multicellular organismwith a requirement for, and a specific transport mechanism for VB12. Forexample, bacteria resistant to a particular antibiotic where theresistance is mediated by the loss of ability to transport theantibiotic inside the cell, could be overcome by this procedure. AVB12-antibiotic complex could thus be effectively delivered inside thebacterial cell via the VB12 transport mechanism. This could lead to anability to reutilize a number of antibiotics whose current use hasbecome limited by development of bacterial resistance. Delivery ofactive substances, of the type described above, could be achieved to aside variety or organisms, particularly parasites of humans or animals.

In another embodiment the invention provides a process for theproduction of a complex comprising, at least one active substance linkedto at least one carrier molecule, said carrier molecule being VB12 or ananalogue thereof, wherein the ability of the carrier to undergo thebinding reactions necessary for uptake and transport of VB12 in avertebrate host and the activity of the active substance aresubstantially maintained which process comprises one or more of thefollowing steps:

a) reacting the active substance with the carrier to form said complex:

b) chemically modifying the active substance to provide at least onefunctional group capable of forming a chemical linkage, and reacting theactive substance and carrier to form said complex;

c) chemically modifying the carrier to provide at least one functionalgroup capable of forming a chemical linkage and reacting the activesubstance and carrier to form said complex;

d) chemically modifying the active substance and the carrier to providefunctional groups capable of forming a chemical linkage, and reactingthe active substance and carrier to form said complex;

e) reacting the active substance with at least one cross-linking agentand reacting the active substance and the carrier molecule to form saidcomplex;

f) reacting the carrier with at least one cross-linking agent andreacting the active substance and carrier to form said complex;

g) reacting the active substance and carrier with at least onecross-linking agent and reacting the active substance and carrier toform said complex.

A preferred process of the invention comprises;

(i) preparing the mono-acid derivative of VB12 by mild acid hydrolysis,and purifying the derivative;

(ii) chemically modifying an active substance to provide at least onefunctional group capable of forming a chemical linkage; and

(iii) reacting the modified active substance and mono-acid derivative ofVB12 to form said complex.

The cross-linking agent may contain a disulfide bond or be cleavable byacid, base or periodate. Examples of cross-linking agents includeN-(4-azidophenylthio)phthalimide, 4,4'-dithlobisphenylazide,dithiobis-(succinimidylpropionate),dimethyl-3,3'-dithiobispropionimidate.2HCl,3,3'-dithiobis-(sulfosuccinimidylpropionate),ethyl-4-azidophenyl-1,4-dithiobutyrimidate, HCl,N-succinimidyl-(4-azidophenyl)-1,3'-dithio-propionate,sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionate,sulfosuccinimidyl-2-(p-azidosalicylamido)-ethyl-1,3'dithiopropionate,N-succinimidyl-3-(2-pyridyldithio)propionate,sulfosuccinimidyl-(4-azidophenyldithio)-propionate, and 2-iminothiolane.Preferred cross-linking agents are disuccinimidyl tartrate and bis-2-(succinimidyloxycarbonyloxy)-ethyl!sulfone.

Suitably, cross-linking of the carrier and active substance may beachieved by acid hydrolysis of the amide groups of the propionamide sidechains adjacent to rings A, B and C of VB12 and coupling to suitablegroups of the active substance.

In a further embodiment of the invention there is provided a medicamentwhich comprises a complex according to the invention together with apharmaceutically acceptable carrier or diluent.

Examples of pharmaceutically acceptable carriers and diluents includetypical carriers and diluents such as sodium bicarbonate solutions andsimilar diluents which neutralize stomach acid or have similar bufferingcapacity, glycols, oils, oil-in-water or water-in-oil emulsions, andinclude medicaments in the form of emulsions, gels, pastes and viscouscolloidal dispersions. The medicament may be presented in capsule,tablet, slow release or elixir form or as a gel or paste. Furthermore,the medicament may be provided as a live stock feed or as food suitablefor human consumption.

The invention also provides an antibacterial formulation comprising acomplex according to the invention, in which the active substance is anantibacterial active substance together with a carrier or diluenttherefor.

In another embodiment the invention provides a method of enhancing ahost vertebrate's response to an orally administered active substancewhich method comprises the oral administration of an effective amount ofsaid active substance as a complex according to the invention, or of amedicament according to the invention.

The invention also provides a method of selectively modulating themagnitude and/or type of immune response to an antigen or hapten, whichmethod comprises orally administering an effective amount of saidantigen or hapten as a complex according to the invention, or of amedicament according to the invention.

The invention also provides a method of delivering an active substanceto any unicellular or multicellular organism, including bacteria,protozoa, or parasites, which has a requirement for VB12 as well as aspecific uptake mechanism for the same, which method comprisesadministering a complex of the invention to the organism. In this mannerbacteria which are resistant to an antibiotic due to the loss of theirability to transport the antibiotic into the cell could be once againmade sensitive to the antibiotic by coupling the antibiotic to VB12 andusing the natural VB12 uptake system of the bacteria to deliver theantibiotic into the cell. In this fashion a number of antibiotics whoseuse has been discontinued due to the occurrence of bacterial resistancecould regain pharmacological significance.

In a further embodiment of the invention there is provided a method ofdelivering an active substance across the blood/brain barrier or acrossthe placenta into a developing foetus by administering a complex of theinvention. Delivery of such substances would occur through the naturalVB12 uptake mechanisms at these barriers.

BEST MODE FOR CARRYING OUT THE INVENTION Materials

Bovine serun albumen (BSA), VB12, p-nitrophenol, LHRH acetate salt, andneomycin sulfate were all purchased from Sigma Chemical Co. St Louis,Mo. USA.

1-ethyl-3-(dimethylaminopropyl)carbodiimide HCl (EDAC) was obtained fromBIORAD Labs, Calif., while N,N'dicyclohexylcarbodiimide (DCC) waspurchased from Fluka.

PREPARATION 1 Monocarboxyl-Derivative of VB

The acid derivative of VB12 can readily be prepared by hydrolysingnative VB12 for 72 h in 0.4M HCl at room temperature. The reaction isstopped by passing the hydrolysate down an ion-exchange column of DOWEXAG1-X8. The flow through containing the monoacid VB12 is lyophilized andresuspended in 0.2M pyridine and adjusted to pH9.05 with 1M ammoniumhydroxide. The solution is then passed down a Sephadex QAE A25previously equilibrated with 0.2M pyridine and the monoacid eludatedwith a gradient from 0.4M pyridine to 0.4M, 0.16M acetic acid. Thefractions containing the purified mono-acid are pooled and lyophilized.

The following examples illustrate preferred embodiments of the inventionand should not be construed as limiting thereon.

The monocarboxyl VB12 can be covalently crosslinked to any aminocontaining compound by the use of a suitable carbodiimide.

EXAMPLE 1 VB12-BSA

VB12-BSA complex was formed by mixing an equal weight of COOH--B12 withBSA in distilled water, the pH was adjusted to 6.5 with 1M NaOH and anequal weight of solid EDAC was added to the solution and allowed toreact overnight. Free, unreacted COOH--B12 was removed by chromatographyof Sephadex G-25, followed by repeated ethanol precipitation of theVB12-BSA complex.

EXAMPLE 2 VB12-Lys-6-LHRH

Monocarboxyl VB12 plus 1.5 equivalents of n-hydroxysuccinamide weredissolved in cold (4° C.) dimethyl formamide (DMF). To this solution wasadded 1.1 equivalents of dicyclohexylcarbodiimide (DCC) in DMF. Thesolutions were warmed to room temperature and allowed to react for 1hour. Lys-6-LHRH dissolved in DMF containing triethylamine was added andallowed to react overnight. The resultant complex was separated from thefree reactants by chromatography on Sephadex G-25 followed by reversephase HPLC.

EXAMPLE 3 VB12-Neomycin

The total acid hydrolisate of VB12 was adjusted to pH6.5 with NaOH, anequal weight of neomycin sulfate was added to the solution followed byan equal weight of EDAC. The conjugation was allowed to proceedovernight after which the conjugate was separated from unreactedreagents by chromatography on G-25 and reverse phase HPLC.

All reactions and purification procedures were monitored by thin layerchromatography. The degree of VB12 substitution of BSA was determined byspectrophotometric scanning of the conjugate using O.D.278 extinctionvalues of 0.6 and 11.5, for 1 mg/ml solutions of BSA and VB12,respectively, and an O.D.361 of 20.4 for VB12.

Female C57B1/6J mice (18-22 g) were obtained from the Animal ResourcesCentre (Perth, Western Australia). All mice received conjugatepreparations in 0.5 ml of 0.1M carbonate/bicarbonate buffer pH9.5 usinga specially prepared feeding needle. Mice were fed on days 0 and 14. Onday 21 the mice were bled from the orbital plexus. Antibody titres ofserum were determined by ELISA using alkaline phosphatase conjugatedanti-mouse serum.

EXAMPLE 4 Stimulation of Serum Antibodies Following Oral Administrationof VB12-BSA Complex

The possible potential for VB12 deliver protein molecules, covalentlylinked to it, from the intestine to the circulation was investigated.The immune response generated to this complex was compared to thatgenerated by the protein fed alone or together with VB12, or to theprotein injected intramuscularly.

As seen in Table 1, feeding mice with microgram quantities of BSA or FGGcoupled to VB12 resulted in the stimulation of significant serumantibody responses to the BSA or FGG respectively. Feeding of eitherprotein in similar amounts or in a 50 fold excess either mixed with VB12or without VB12 resulted in the stimulation of no anti-BSA or anti-FGGantibodies. Feeding of these VB12-protein complexed was also capable ofstimulating good cellular immunity (as measured by the footpad assay forDTH).

                  TABLE 1                                                         ______________________________________                                        Immune response to orally presented VB12-BSA or VB12-FGG complex                             Serum Antibody                                                                            Footpad                                            Oral Immunogen Response *  Response +                                         ______________________________________                                        BSA (50 μg) <4          0                                                  BSA (2500 μg)                                                                             <4          nd                                                 VB12           <4          0                                                  VB12 + BSA     <4          0                                                  VB12 - BSA     1351 ± 198                                                                             17.3 ± 5                                        FGG            <4          0                                                  VB12 + FGG     <4          0                                                  VB12 - FGG     1584 ± 647                                                                             23.3 ± 6                                        FGG + FCA s.c. 16504 ± 3047                                                                           27.4 ± 4                                        ______________________________________                                         * The reciprocal of the antiserum dilution that gave an ELISA reading of      0.5 afer 45min. at 37° C. on day 21 after initial feeding. Each        value represents the mean of 15 mice ±1 standard deviation. Mice           received two feedings of antigen (50 μg) on days 1 and 14. On day 21       mice were bled from the retro orbital plaxus and the antibody titres          measured by ELISA as described previously (RussellJones et al., 1984).        Each protein molecule was substituted with an average of 5 VB12 groups.       + Footpad swelling was measured in mm using a microcaliper. All groups        received a 50 μg priming dose of antigen followed by challenge with 10     μg of the immunizing antigen in the right foot and 10 μg of             ovalbumen in the left footpad. Swelling was measured after 24 h.         

EXAMPLE 5 Oral Delivery of VB12-LHRH as a Means of Stimulating Ovulation

Although a number of hormones as oestrogen and progesterone are activelyabsorbed upon oral administration, there are may other which have littleeffect when given per os. Noteable amongst these hormones is the peptidehormone lutenizing hormone releasing hormone (LHRH), or gonadotrophinreleasing hormone (GnRH). This hormone is normally secreted by theanterior pituitary and is responsible for the control of release oflutenizng hormone (LH) and follicle stimulating hormone (FSH).Parenteral injections of LHRH have previously been shown to be effectivein stimulating FSH and LH release, however orally presented LHRH haslittle effect. Many studies have been performed on varying the sequenceof LHRH, with the result that a number of agonists and antagonists havenow been identified. Perhaps one of the most powerful agonistsidentified to date is the D-Lys-6 analogue of LHRH (D-Lys-6.LHRH). Asthe epsilon amino group on the lysine of this analogue is readilyaccessible for peptide cross-linking it was decided to use the DCCmethod to link morocarboxyl VB12 to D-Lys6.LHRH and to test it'sefficacy upon oral administration.

The D-lys-6 analogue of LHRH was synthesized by us and purified byreverse phase HPLC. The purified analogue was coupled to monocarboxylVB12 using DCC as described in Example 2. The conjugated product waspurified by Sephadex G-25 chromatography in 10% acetic acid, followed byHPLC Chromatography.

Mature C57B1/6J female mice were treated in the following fashion: Onday 0 all mice received a subcutaneous (s.'c) superovulating dose ofpregnant mare serum gonadotraphin (PMSG) to stimulate the growth ofovarian follicles. After 48 hours mice received various doses of LHRH,Lys-6-LHRH or saline. On day 3 mice were sacrificed and examined forovulation. Ovulation was assessed by examining for the presence ofcorpora haemorrhagica on the ovaries using a stereoscopic microscope at80×0 power.

The results below show that by coupling Lys-6-LRH to VB12 it is possibleto deliver the analogue orally and to still observe a biological effectas exemplified by it's ability to stimulate ovulation in developingfollicles. The inability of this preparation to exert it's effect wheninjected intravenously presumably reflects the rapid clearance of freeVB12 when it is not complexed to transcobalamin II.

                  TABLE 2                                                         ______________________________________                                        Demonstration of the biological activity of Lys-6-LHRH                        Treatment                   Number of mice                                    Day 0      Day 2            ovulating                                         ______________________________________                                        PMSG 8 IU  Lys-6-LHRH 50     μg iv                                                                           3/4                                         PMSG 8 IU  LHRH       50     μg iv                                                                           1/4                                         PMSG 8 IU  Saline     250    μl iv                                                                           0/4                                         PMSG 4 IU  Lys-6-LHRH 50     μg iv                                                                           3/3                                         PMSG 4 IU  LHRH       50     μg iv                                                                           1/3                                         PMSG 4 IU  Saline     250    μl iv                                                                           0/3                                         ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Demonstration of the ability of VB12 to deliver the Lys-6-LRH orally          Treatment                   Number of mice                                    Day 0    Day 2              ovulating                                         ______________________________________                                        PMSG 8 IU                                                                              VB12-Lys-6-LHRH                                                                            50     μg iv                                                                           0/5                                         PMSG 8 IU                                                                              VB12-Lys-6-LHRH                                                                            50     μg/os                                                                           3/5                                         PMSG 8 IU                                                                              LHRH         50     μg/os                                                                           1/5                                         PMSG 8 IU                                                                              Saline       250    μl/os                                                                           0/5                                         ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Dose response to orally presented VB12-Lys-6-LHRH                             Treatment                   Number of mice                                    Day 0    Day 2              ovulating                                         ______________________________________                                        PMSG 8 IU                                                                              VB12-Lys-6-LHRH                                                                            50     μg/os                                                                           4/5                                         PMSG 8 IU                                                                              VB12-Lys-6-LHRH                                                                            25     μg/os                                                                           3/5                                         PMSG 8 IU                                                                              VB12-Lys-6-LHRH                                                                            12     μg/os                                                                           5/5                                         PMSG 8 IU                                                                              VB12-Lys-6-LHRH                                                                            6      μg/os                                                                           2/5                                         PMSG 8 IU                                                                              LHRH         50     μg/os                                                                           1/5                                         PMSG 8 IU                                                                              LHRH         25     μg/os                                                                           1/5                                         PMSG 8 IU                                                                              LHRH         12     μg/os                                                                           0/5                                         PMSG 8 IU                                                                              LHRH         6      μg/os                                                                           0/5                                         PMSG 8 IU                                                                              HCG          10     IU iv                                                                              5/5                                         PMSG 8 IU                                                                              Saline       250    μl/os                                                                           0/5                                         ______________________________________                                    

EXAMPLE 6

A number of drugs including the antibiotic, neomycin, are highlyeffective antibiotics when injected parenterally, however they arecompletely ineffective when given orally as they cannot be transportedacross the intestinal epithelium. It was therefore decided to see ifVB12 could act as a carrier for an antibiotic (neomycin) which normallyhas no effect upon a systemic infection when the antibiotic was givenorally.

Neomycin was covalently linked to VB12 as described in Example 3 and fedto mice infected with S. typhimurium.

Oral administration of neomycin, or neomycin plus VB12 was not able toeliminate systemic infection with S. typhimurium. When neomycin wascoupled to VB12, however, a significant quantity of the conjugate wastransported across the intestinal epithelium and was capable ofeliminating a systemic Salmonella infection. Table 3 shows that miceinfected with S. typhimurium could be saved by either feedingVB12.neomycin conjugate (1 mg total dose) or by the i.m. injection ofneomycin or VB12.neomycin (both 1 mg total dose). All other treatmentsfailed to prevent death due to infection. In addition, the extent towhich orally presented VB12.neomycin was capable of clearing infectiveparticles from the liver and spleen of experimental animals suggeststhat, at least for this dosage, VB12.neomycin is comparable to and ani.m. injection of neomycin alone or the neomycin.VB12 conjugate (Table5).

                  TABLE 5                                                         ______________________________________                                        Bactericidal properties of VB12-neomycin conjugates                                             Suvivors (day 10)                                           Treatment    Route      Number  Percentage                                    ______________________________________                                        Saline       oral       0       0                                             Neomycin     oral       0       0                                             VB12         oral       0       0                                             Neomycin + VB12                                                                            oral       0       0                                             Neomycin - VB12                                                                            oral       2       100                                           Saline       i.m.       0       0                                             Neomycin     i.m.       2       100                                           Neomycin - VB12                                                                            i.m.       2       100                                           ______________________________________                                    

Male C57B1/6J mice (/group) were fed 1×10⁶ S. typhimurium on day 0. Onday 3 mice received either saline, VB12, VB12+neomycin (Neomycin+VB12),VB12 coupled to neomycin (Neomycin-VB12), or neomycin alone. A totaldose of 1 mg was administered as five smaller doses each separated by 12hours. Neomycin was coupled to VB12 and the conjugate purified asoutlined in Example 3.

It is possible to covalently couple VB12 to proteins (FGG and BSA),hormones (LHRH) and antibiotics (neomycin) and to utilize the naturalactive uptake mechanism for VB12 to transport these molecules from thelumen of the gut into the systemic circulation while retaining fullimmunogenicity and/or biological activity of the molecules coupled toVB12. The importance of these findings lie in the potential use of VB12as a specific carrier of highly potent hormones, antibiotics andvasoactive peptides which currently must be repeatedly administered byinjection at considerable costs and inconvenience.

INDUSTRIAL APPLICABILITY

The present invention provides a simple and novel technique for thespecific oral presentation of various molecules previously incapable ofbeing transported across the gut in significant amounts or in producinga significant systemic immune response upon oral feeding of variousantigens. These antigens would not normally elicit an immune responsewhen fed unless very large quantities of antigen were administered.Similarly various active molecules which are normally only poorlyabsorbed from the intestine can be covalently linked to VB12 and sorender them susceptable to intestinal uptake.

REFERENCES

1. Castle, W. B. N. Engl. J. Med, 24, 603-611 (1953)

2. Fox, H. J., Castle, W. B. Am. J. Med. Sci., 203, 18-26

3. Hoedemaeker, P. J., Ables J., Wachters, J. J. Averds, A., Nieweg,H.O., Lab. Invest., 15, 1163-1169 (1966)

4. Allen, R. H. Majerus, P. W. J. Biol. Chem.,; 247, 7702-7708 (1972)

5. Allen, R. H. Majerus, P. W. J. Biol. Chem.,; 247, 7709-7717 (1972)

6. Grasbech, R. Progr. Haematol. 6, 233-260 (1969)

7. Allen, R. H. Majerus, P. W. J. Biol. Chem.,; 247, 7695-7701 (1972)

8. Russel-Jones, G. J., Gotschlich, E. C. Blake, M. S. J. Exp. Med.,160, 1476-(1984)

9. Sedgwick, J. D. Holt, P. G. J. Immunol. Meth., 87 37-44 (1986)

We claim:
 1. A method of treating a patient in need of treatment with a biologically active substance selected from the group consisting of a protein, a peptide, a hormone, and a polysaccharide, comprising the steps of(1) providing an orally administrable complex comprising said biologically active substance covalently linked to a vitamin B12 carrier molecule, wherein said carrier molecule is capable of binding in vivo to intrinsic factor, thereby enabling uptake and transport of the complex from the intestinal lumen of said patient via intrinsic factor to the systemic circulation of said patient, and (2) orally administering said complex to said patient so as to elicit a physiological effect associated with the presence of said biologically active substance in the systemic circulation of said patient.
 2. The method according to claim 1, wherein said biologically active substance is a hormone selected from the group consisting of luteinizing hormone releasing hormone, insulin, testosterone, pregnant mare serum gonadotrophin, human chorionic gonadotrophin and inhibin.
 3. The method according to claim 2, wherein said hormone is the lys-6 form of LHRH.
 4. The method according to claim 1, wherein said biologically active substance is a protein, peptide, or polysaccharide antigen or hapten,wherein said antigen or hapten is selected from the group consisting of grass pollen, weed pollen, tree pollen, plant pollen, cat hair, dog hair, pig hair, or other epithelia, house dust mite, wheat chaff, and kapok antigens or haptens, or wherein said antigen or hapten is selected from the group consisting of a protein from or immunogens against influenza, measles, Rubella, smallpox, yellow fever, diphtheria, tetanus, cholera, plague, typhus, BCG, tuberculosis causing agents, Haemophilus influenza, Neisseria catarrhalis, Klebsiella pneumoniae, pneumococci, streptococci, a malarial parasite and a causative agent of coccidiosis in chickens, or wherein said antigen or hapten is a secretory product from an organism selected from the group consisting of diphtheria, tetanus, cholera, plague, typhus, and tuberculosis causing agents, Haemophilus influenza, Neisseria catarrhalis, Klebsiella pneumoniae, pneumococci, and streptococcus mutans.
 5. A method of treating a patient in need of treatment with a biologically active substance, comprising the steps of(1) providing an orally administrable complex comprising said biologically active substance covalently linked to a vitamin B12 carrier molecule, wherein said carrier molecule is capable of binding in vivo to intrinsic factor, thereby enabling uptake and transport of the complex from the intestinal lumen of said patient via intrinsic factor to the systemic circulation of said patient, and (2) orally administering said complex to said patient so as to elicit a physiological effect associated with the presence of said biologically active substance in the systemic circulation of said patient, wherein said biologically active substance is a therapeutic agent selected from the group consisting of neomycin, salbutamol, pyrimethamine, penicillin G, methicillin, carbenicillin, pethidine, xylazine, ketamine hydrochloride, mephenesin and iron dextran.
 6. The method according to claim 1, wherein said vitamin B12 carrier molecule is selected from the group consisting of cyanocobalamin, aquocobalamin, adenosylcobalamin, methylcobalamin, hydroxycobalamin, cyanocobalamin carbanilide, 5-0-methylbenzylcobalamin, desdimethyl, monoethylamide and methylamide analogues of cyanocobalamin, aquocobalamin, adenosylcobalamin, methylcobalamin, hydroxycobalamin, cyanocobalamin carbanilide and 5-0-methylbenzylcobalamin, coenzyme B12, 5'-deoxyadenosyl-cobalamin, chlorocobalamin, sulphitocobalamin, nitrocobalamin, thiocyanatocobalamin, adenosylcyanocobalamin, cobalamin lactone, cobalamin lactam, vitamin B12 anilide, vitamin B12 propionamide, and a vitamin B12 molecule in which one or two corrin ring side chains are free carboxylic acids.
 7. The method according to claim 1, wherein said vitamin B12 carrier molecule includes a central metal atom selected from the group consisting of Ni and Zn.
 8. The method according to claim 1, wherein said vitamin B12 carrier molecule is a cyanocobalamin methylamide or a cobalamin ethylamide.
 9. The method according to claim 1, wherein said vitamin B12 carrier molecule is selected from the group consisting of 5,6-dichlorobenzimidazolecobalamin, 5-hydroxybenzimidazole-cobalamin, and trimethylbenzimidazolecobalamin.
 10. The method according to claim 1, wherein said biologically active substance is covalently linked via a cross-linking agent to a vitamin B12 carrier molecule and said cross-linking agent is selected from the group consisting of a N-(4-azidophenylthio)phthalimide, 4,4'-dithiobis-phenylazide, dithiobis(succinimidylpropionate), dimethyl-3,3'-dithiobispropionimidate.2HCl, 3, 3 '-dithiobis(sulphosuccinimidyl-propionate), ethyl-4-azidophenyl-1, 4-dithiobutyrimidate.HCl, N-succinimidyl-(4-azidophenyl)-1,3'-dithiopropionate, sulphosuccinimidyl-2-(p-azidosalicylamido)-ethyl-1,3'-dithiopropionate, N-succinimidyl-3-(2-pyridyldithio)propionate, sulphosuccinimidyl-(4-azidophenyldithio)-propionate and 2-iminothiolane.
 11. The method according to claim 10, wherein said cross-linking agent is bis(2-(succinimidyloxycarbonyloxy)-ethyl)sulphone.
 12. The method according to claim 1 wherein said biologically active substance is administered orally in a formulation comprising(i) an orally administrable complex comprising said biologically active substance covalently linked via a cross-linking agent to a vitamin B12 carrier molecule, wherein(A) said carrier molecule is capable of binding in vivo to intrinsic factor, thereby enabling uptake and transport of the complex from the intestinal lumen of said patient via intrinsic factor to the systemic circulation of said patient, and (B) said cross-linking agent links said biologically active substance to a carboxyl group of an acid-hydrolyzed propionamide side chain adjacent to ring A, ring B or ring C of said carrier molecule;and (ii) an orally and pharmaceutically acceptable carrier or diluent.
 13. The method according to claim 12, wherein said formulation is in an oral delivery form selected from the group consisting of a capsule, a tablet, an emulsion, a viscous colloidal dispersion, an elixir, a gel and a paste. 